Comprehensive measurements of reactivities and inhibitory activities of various medicaments for drug-metabolizing enzymes are very important for predicting onset of side effects, medicament interactions and the like induced by administration of the medicaments. For example, cytochrome P-450 is a typical drug-metabolizing enzyme, and since a medicament having an inhibitory action on cytochrome P-450 may produce side effects, high throughput screening (HTS) systems have been developed, which enable efficient measurement of inhibitory activities of various medicaments against cytochrome P-450. However, there are also medicaments which are detoxified by UDP-glucuronosyltransferase, which is a conjugation enzyme (henceforth also abbreviated as “UGT” in this specification), in the metabolic processes of the medicaments, and there are many cases where a drug-metabolizing enzyme other than cytochrome P-450 plays a more important role.
UGT is a kind of phase II conjugation reaction enzymes, and metabolizes endogenous substances such as bilirubin and steroid hormones and exogenous substances such as medicaments, carcinogens and environmental pollutants as substrates, and responsible for about 15% of the metabolism of major medicaments. And there have been made reports about a possibility that elimination kinetics of medicaments such as acetaminophen, lamotrigine and lorazepam are changed by variation of the metabolic activity of UTG based on genetic polymorphism of UGT isozymes. Among them, a research that most definitely demonstrates the influence of the UGT gene polymorphism includes the research on the gene polymorphism of UGT1A1, a kind of the UGT isozyme responsible for metabolism and detoxification of the active metabolite SN-38 of irinotecan hydrochloride (topoisomerase I inhibitor) which is used for the therapeutic treatment of solid tumors such as metastatic colon cancer (Isomura, M., et al., Gan To Kagaku Ryoho., 32, 1908, 2005).
Irinotecan hydrochloride, which is a prodrug, is converted into the active type, SN-38, by carboxy esterase, and then SN-38 undergoes glucuronidation by UGT1A1 and thereby detoxified into the inactive type in vivo. However, in patients with low UGT1A1 activity, the conversion from the active type into the inactive type is insufficient, thus SN-38 stays in the body for a long period of time, and therefore risk associated with onset of side effects increases. Where a medicament to be detoxified by UGT such as irinotecan hydrochloride is administered, this result also suggests a possibility that more critical side effects may be induced by UGT1A1 inhibition caused by the use of other combined medicaments. Therefore, in recent years, UGT has attracted much attention as a drug-metabolizing enzyme, and especially UGT1A1 isozymes has been focused.
From the viewpoint mentioned above, it is necessary, in the screening of candidates for drug development, to investigate UGT inhibitory action of the candidate compounds for predicting onset of side effects. However, the currently available test methods are quantitative analysis methods using purification apparatuses such as HPLC, and thus they have a problem in that they are inconvenient and time consuming. Therefore, it is desired to develop an HTS system which enables simple and short time measurement of UGT inhibitory activity of lots of test compounds. In particular, there is desired an HTS system which enables convenient and rapid measurement of UGT inhibitory activity by using a fluorescent probe with which the UGT activity can be highly sensitively measured.
Scopoletin having the coumarin structure was found in recent years as a probe for detecting the UGT activity by fluorescence (http://www.bdbiosciences.com/discovery_labware/gentest/products/pdf/). However, this probe is a short wavelength excitation type probe, which emits fluorescence with an excitation light of around 300 nm, and thereby because the probe is easily influenced by intracellular background fluorescence, the probe has a problem in that it is inapplicable to tests using cells or tissues. The probe also has a problem in that its sensitivity is low due to its low molar absorption coefficient.